EP3724378A1 - Yeast display of proteins in the periplasmic space - Google Patents
Yeast display of proteins in the periplasmic spaceInfo
- Publication number
- EP3724378A1 EP3724378A1 EP18889782.1A EP18889782A EP3724378A1 EP 3724378 A1 EP3724378 A1 EP 3724378A1 EP 18889782 A EP18889782 A EP 18889782A EP 3724378 A1 EP3724378 A1 EP 3724378A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- yeast
- antibody
- yeast host
- periplasm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/75—Agonist effect on antigen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
- C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation
- C07K2319/912—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation containing a GPI (phosphatidyl-inositol glycane) anchor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the disclosure relates to cell display and methods of high-throughput screening of protein libraries.
- the disclosure relates to methods for displaying proteins in the periplasmic space of yeast and the use of such methods for screening protein libraries for specific binding or functional characteristics.
- Molecular display technology has proven invaluable for the discovery, production, and optimization of proteins and peptides for a variety of biotechnological and biomedical applications.
- Various approaches including phage display (Smith (1985) Science 228: 1315- 1317), mRNA (Wilson et al. (2001) Proc. Natl. Acad. Sci. USA 98:3750-3755) and DNA display (Yonezawa et al. (2003) Nucleic Acids Res. 31 :e 118), ribosome display (Hanes & Pluckthun (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942), eukaryotic virus display (Bupp & Roth (2002) Mol. Ther.
- Biotechnol. 15:553-557 have been developed to screen combinatorial libraries of recombinant proteins for desired characteristics.
- Such display technologies have been widely used in protein engineering to identify proteins having improved stability and desired binding affinities and enzymatic activities, and have found use in various applications, including directed evolution, affinity maturation, therapeutic protein and antibody engineering, biofuel production, adsorption of environmental pollutants, epitope mapping, and study of protein-protein interactions.
- yeast display has been used to display a wide variety of prokaryotic and eukaryotic proteins (Cherf et al. (2015) Methods Mol. Biol. 1319: 155-175). Expression in yeast cells provides the advantage of allowing proper folding and glycosylation of eukaryotic proteins. In conventional yeast display, recombinant proteins are displayed on the surface of yeast cells by fusion to a cell wall protein.
- Saccharomyces cerevisiae has been the most commonly used species for cell surface display
- other yeast species including Pichia, Candida, and Yarrowia strains have found use for some applications (Tanaka et al. (2012) Appl. Microbiol. Biotechnol.
- the present disclosure relates to high-throughput screening of protein libraries for specific binding or functional characteristics by displaying proteins in the periplasmic space of yeast cells.
- the invention includes a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises: a) a protein variant for display in the yeast host cell periplasmic space, wherein the displayed protein variant is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of protein variants; b) a periplasm anchor protein, wherein the periplasm anchor protein is linked to the protein variant such that the protein variant is displayed in the periplasmic space; and c) a target membrane protein of interest, wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the protein variant displayed in the yeast host cell periplasmic space.
- the yeast host cells may be haploid or diploid.
- the protein variant and the periplasm anchor protein are covalently linked together in a fusion protein.
- the protein variant and the periplasm anchor protein are noncovalently linked together by molecular binding interactions in a complex.
- the protein variant and the periplasm anchor protein are linced by a linked by a non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- the periplasm anchor protein comprises a signal sequence that directs transport of the fusion protein to the yeast host cell periplasm, plasma membrane, or cell wall such that the fused protein variant is displayed in the periplasm.
- An exemplary signal sequence that can be used is the prepro-alpha-factor signal sequence.
- the periplasm anchor protein comprises a membrane-spanning transmembrane domain that projects the fused protein variant into the periplasm.
- the periplasm anchor protein comprises a cell-membrane associated protein domain that localizes to an external face of the cell membrane such that the displayed protein variant is projected into the periplasm.
- the cell- membrane associated protein domain is a glycosylphosphatidylinositol (GPI)-plasma membrane anchoring domain.
- GPI-plasma membrane anchoring domain may be a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- the periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the displayed protein variant is projected into the periplasm.
- the periplasm anchor protein comprises a signal sequence that directs transport of the fusion protein to the yeast host cell periplasm, and the periplasm anchor protein is sufficiently large that the fusion protein is retained in the periplasm.
- the anchor protein is a component of a periplasmic protein complex that is sufficiently large that formation of the complex in the periplasm results in retention of the fusion protein in the periplasm.
- the fusion protein further comprises a tag.
- the protein variants are antibodies, antibody mimetics, aptamers, antigens, enzymes, receptors, hormones, substrates, agonists, antagonists, or ligands.
- the protein variants are antibodies selected from the group consisting of monoclonal antibodies, chimeric antibodies, nanobodies, recombinant fragments of antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, F v fragments, and scFv fragments.
- each yeast host cell in the yeast periplasmic display library further comprises a target protein of interest that is expressed in a location accessible to the displayed protein variant (e.g., in close enough proximity for the displayed protein variant to bind to the target protein of interest).
- the target protein of interest may be located in the yeast host cell plasma membrane or periplasm.
- the target protein of interest can be, for example, a membrane protein, a receptor, an ion channel, or a transporter.
- the target protein of interest is a G-protein coupled receptor (GPCR).
- GPCR G-protein coupled receptor
- each yeast host cell further comprises a reporter system for detecting a response of the target protein of interest to a protein-protein interaction with the displayed protein variant.
- the displayed protein variant is an antagonist of the target protein of interest, and the response is a decrease in activity of the target protein of interest upon binding of the antagonist to the target protein of interest, wherein the reporter system detects the decrease in activity of the target protein of interest upon binding of the antagonist to the target protein of interest.
- the displayed protein variant is an agonist of the target protein of interest, and the response is an increase in activity of the target protein of interest upon binding of the agonist to the target protein of interest, wherein the reporter system detects the increase in activity of the target protein of interest upon binding of the agonist to the target protein of interest.
- the yeast periplasmic display library may be screened for an agonist of the target protein of interest by culturing at least a subset of the yeast host cells of the yeast periplasmic display library in a media, wherein growth of a yeast host cell in the media indicates that the protein variant displayed in the yeast host cell is an agonist of the target protein of interest.
- activation of the target protein of interest decreases growth of the yeast host cells.
- the yeast periplasmic display library may be screened for an antagonist of the target protein of interest by culturing at least a subset of the yeast host cells of the yeast periplasmic display library in a media, wherein growth of a yeast host cell in the media indicates that the protein variant displayed in the yeast host cell is an antagonist of the target protein of interest.
- the invention includes a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises: a) a fusion protein comprising a periplasm anchor protein fused to an antibody for display in the yeast host cell periplasmic space, wherein the displayed antibody is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of antibodies; and b) a target membrane protein of interest, wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- the target membrane protein of interest may be, for example, a receptor, an ion channel, and a transporter.
- the target membrane protein of interest comprises a mutation that increases or decreases its activity.
- Antibodies that may be displayed with the target membrane protein of interest may include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, recombinant fragments of antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, F v fragments, and scFv fragments.
- the yeast periplasmic display library further comprises a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target membrane protein of interest is activated to allow detection of increases or decreases in activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest.
- the reporter gene may be a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), antibiotic resistance marker (e.g., confers resistance to an antibiotic such as geneticin (e.g., aphAl), zeocin (e.g., ble), hygromycin B, nourseothricin, or bialaphos), fluorescent marker (e.g., of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein), biolumine scent marker (e.g., luciferase or aequorin), or counter-selectable marker (e.g., CAN 1, URA3, MET15, TRP1, and TK).
- antibiotic resistance marker e.g., confers resistance to an antibiotic such as geneticin (e.g., aphAl), zeocin (e.g., ble),
- the reporter gene is a selectable marker such that increases in activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest are detectable by growth of the yeast host cells on a positive selection media.
- the reporter gene is a counter-selectable marker such that decreases in activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest are detectable by growth of the yeast host cells on media comprising a counterselection agent.
- the target membrane protein of interest is a G-protein coupled receptor (GPCR), for example, an exogenous GPCR such as a mammalian GPCR (e.g., from human or nonhuman primate, rodent, laboratory animal, livestock).
- GPCR G-protein coupled receptor
- the mammalian GPCR is a human GPCR selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR, and AT1R.
- the yeast periplasmic display library further comprises an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- the yeast Ga subunit may belong to a Gai, Gaq, Gas, or Gao family G protein.
- the chimeric Ga subunit at least five C-terminal residues of a yeast Ga subunit may be replaced with corresponding C-terminal residues of a mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by a mammalian GPCR.
- the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR.
- the chimeric Ga subunit comprises at least 41 N-terminal residues of the yeast Ga subunit.
- Exemplary mammalian Ga subunits include G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q, G alpha-l 1, G alpha-l2, G alpha-l3, and transducin.
- the target GPCR of interest has constitutive ligand-independent activity.
- a ligand is added to activate the target GPCR of interest.
- the yeast host cell is a haploid or diploid yeast host cell. In certain embodiments, the yeast host cell is a ⁇ far 1. ⁇ sst2. ⁇ stc 14. ⁇ stc3 or ⁇ mat strain.
- a ⁇ mat strain may comprise, for example, a deleted or inactivated MATa locus or a deleted or inactivated MATa locus.
- the yeast host cell further comprises a modified CLN3 protein comprising a C-terminal truncation that increases abundance of CLN3 in the yeast host cell compared to a wild-type CLN3 protein.
- the modified CLN3 protein may retain at least N-terminal amino acids 1-387 or 1-408 of the wild-type CLN3 protein, or any number of N- terminal amino acids within these ranges, such as 1-388, 1-389, 1-390, 1-391, 1-392, 1-393, 1- 394, 1-395, 1-396, 1-397, 1-398, 1-399, 1-400, 1-401, 1-402, 1-403, 1-404, 1-405, 1-406, 1-407, or 1-408, wherein the C-terminal truncation comprises a deletion of all or some of the remaining residues of the wild-type CLN3 protein.
- the yeast host cell is a FAR1 strain for selection of antibody antagonists of
- the yeast host cell is a ⁇ farl strain comprising a pheromone- inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of a GPCR.
- the invention provides a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises: a) an antibody for display in the yeast host cell periplasmic space, wherein the displayed antibody is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of antibodies, wherein the antibody is linked to a signal sequence that directs transport of the antibody to the yeast host cell periplasm, plasma membrane or cell wall, such that the antibody is displayed in the yeast host cell periplasmic space; and b) a target membrane protein of interest, wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- the invention includes a method of making a yeast periplasmic display library described herein, the method comprising: a) providing a plurality of recombinant polynucleotides encoding fusion proteins, wherein each recombinant polynucleotide encodes a different fusion protein comprising the periplasm anchor protein fused to a different antibody for display; b) transfecting the plurality of yeast host cells with the plurality of recombinant polynucleotides encoding the fusion proteins; c) transfecting the plurality of yeast host cells with a recombinant polynucleotide encoding the target membrane protein of interest; and d) culturing the plurality of yeast host cells under conditions that permit expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest localizes to the plasma membrane (i.e., where it is accessible to
- the recombinant polynucleotides encoding the fusion proteins or the recombinant polynucleotide encoding the target membrane protein of interest are provided by expression vectors. In other embodiments, the recombinant polynucleotides encoding the fusion proteins or the target membrane protein of interest are integrated into the yeast host cell genome at a target locus.
- the invention provides a method of making the yeast periplasmic display library, the method comprising: a) providing a first plurality of recombinant polynucleotides encoding the antibodies for display in the yeast host cell periplasmic space, wherein the displayed antibody is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of antibodies; b) providing a second recombinant polynucleotide encoding the periplasm anchor protein, wherein the periplasm anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space; c) transfecting the plurality of yeast host cells with the first plurality of recombinant polynucleotides and the second recombinant polynucleotide; d) transfecting the plurality of yeast host cells with a recombinant polynucleotide encoding the target membrane protein of interest; and e) culturing the plurality of yeast host cells
- each recombinant polynucleotide comprises a promoter operably linked to a polynucleotide encoding a fusion protein or a target membrane protein of interest.
- the recombinant polynucleotide may be provided by a vector comprising the promoter.
- a chromosomal target locus comprises a promoter that becomes operably linked to a polynucleotide encoding a fusion protein or a target membrane protein of interest that integrates at a chromosomal target locus.
- the method further comprises introducing into the plurality of yeast host cells a recombinant polynucleotide encoding an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the invention includes a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a polynucleotide encoding a protein variant after the polynucleotide encoding the signal peptide; c) a polynucleotide encoding a glycophosphatidylinositol (GPI) plasma membrane anchoring domain, positioned such that the vector is capable of producing a fusion protein comprising the signal peptide and the protein variant fused to the GPI plasma membrane anchoring domain; and d) a promoter operably linked to sequences encoding the fusion protein.
- a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a polynucleotide en
- the signal peptide comprises a prepro-alpha-factor signal sequence.
- the cloning site comprises one or more restriction sites.
- the GPI plasma membrane anchoring domain is a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS 1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- the periplasm targeting expression vector further comprises a polynucleotide encoding a linker, wherein said polynucleotide encoding the linker is positioned in between the cloning site and the
- the linker may further comprise a tag.
- the periplasm-targeting expression vector further comprises a selectable marker.
- the invention includes a method of making a yeast periplasmic display library described herein, the method comprising: a) providing a plurality of recombinant polynucleotides encoding antibody variants, wherein each recombinant polynucleotide encodes a different antibody variant; b) transfecting the plurality of yeast host cells with a periplasm targeting expression vector described herein c) transfecting the plurality of yeast host cells with the plurality of recombinant polynucleotides encoding the antibody variants, wherein in each yeast host cell, a recombinant polynucleotide encoding an antibody variant is integrated into the cloning site of the periplasm-targeting expression vector by homologous recombination to allow expression of a fusion protein comprising a periplasm anchor protein fused to an antibody variant for display; c) transfecting the plurality of yeast host cells with a recombinant polynu
- the method further comprises introducing into the plurality of yeast host cells a recombinant polynucleotide encoding an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the invention includes a method of screening a yeast periplasmic display library comprising a reporter system, as described herein, for an antibody that modulates activity of the target membrane protein of interest, the method comprising culturing at least a subset of the yeast host cells of a yeast periplasmic display library described herein in a selection media; and detecting expression of the reporter gene, wherein increased expression of a reporter gene indicates that the antibody increases activity of target membrane protein of interest and decreased expression of the reporter gene indicates that the antibody decreases activity of the target membrane protein of interest.
- Exemplary reporter genes include a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), an antibiotic resistance marker (e.g., confers resistance to an antibiotic such as geneticin (aphAl), zeocin (ble), hygromycin B, nourseothricin, and bialaphos), a fluorescent marker (e.g., of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein), bioluminescent marker (e.g., luciferase or aequorin), and a counter-selectable marker (e.g.,
- the method further comprises positive selection for expression of a nutritional marker, wherein growth of the yeast host cells in a nutrient-deficient selection media indicates the target membrane protein of interest is activated.
- the method further comprises positive selection for expression of an antibiotic resistance marker, wherein growth of the yeast host cells in a selection media comprising an antibiotic indicates the target membrane protein of interest is activated.
- the method further comprises positive selection for expression of a fluorescent marker, wherein detection of fluorescence emitted by the yeast host cells indicates the target membrane protein of interest is activated.
- the method further comprises positive selection for expression of a bioluminescent marker, wherein detection of bioluminescence emitted by the yeast host cells indicates the target membrane protein of interest is activated.
- the method further comprises negative selection for expression of the counter-selectable marker, wherein decreases in activity of the target membrane protein of interest upon binding of the displayed antibody to the target membrane protein of interest are detectable by growth of the yeast host cells in a media comprising an agent that selects against cells expressing the counter-selectable marker.
- the invention includes a method of screening a yeast periplasmic display library for an antibody that modulates the activity of a target GPCR of interest, the method comprising culturing at least a subset of the yeast host cells of the yeast periplasmic display library in a media, wherein detection of activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell not having an antibody displayed in the periplasmic space indicates that the displayed antibody in said at least one yeast host cell binds to and modulates the activity of the GPCR.
- the method further comprises contacting the human GPCR with a ligand.
- the GPCR has constitutive ligand-independent activity.
- the yeast host cell comprises an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- the yeast Ga subunit may belong to a Gai, Gaq, Gas, or Gao family G protein.
- the chimeric Ga subunit at least five C-terminal residues of a yeast Ga subunit may be replaced with corresponding C-terminal residues of a mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by a mammalian GPCR. In some embodiments, at least 20 C- terminal residues of the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR. In another embodiment, the chimeric Ga subunit comprises at least 41 N- terminal residues of the yeast Ga subunit.
- Exemplary mammalian Ga subunits include G alpha- S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q, G alpha-l 1, G alpha-l2, G alpha-l3, and transducin.
- the yeast host cell is a FAR1 strain, wherein inhibition of the pheromone response by an antibody acting as an antagonist that binds to an inhibits the GPCR in the yeast host cell results in cessation of cell cycle arrest and growth of the yeast host cell.
- the yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds to and activates the GPCR in the yeast host cell results in increased expression of the reporter gene.
- the invention provides a yeast host cell comprising: a) an antibody for display in the yeast host cell periplasmic space, b) a periplasm anchor protein, wherein the periplasm anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space; and c) a target membrane protein of interest, wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- the invention provides an antibody linked to a a periplasm anchor protein.
- the the antibody is produced in a yeast host cell, the antibody is localized to the yeast host cell periplasmic space.
- the antibody and the periplasm anchor protein are noncovalently linked together by molecular binding interactions in a complex or are linked by a covalent non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- the antibody and the periplasm anchor protein are covalently linked together in a fusion protein.
- the invention provides a method of localizing an antibody to a yeast host cell periplasmic space comprising linking the antibody to a periplasm anchor protein such that the antibody is localized to the periplasmic space.
- the antibody and the periplasm anchor protein are noncovalently linked together by molecular binding interactions in a complex or are linked by a covalent non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- the antibody and the periplasm anchor protein are covalently linked together in a fusion protein.
- the invention includes a kit comprising a yeast periplasmic display library described herein and instructions for screening a plurality of protein variants for their ability to bind and/or modulate activity of a target protein of interest.
- FIGS. 1A-1D show novel yeast cell display method for screening for antibodies that modulate the function of GPCRs.
- FIG. 1A shows the unique combination of 1) functional human GPCR-yeast coupling to 2) affinity molecule secretion and 3) affinity molecule localization, together in a high-throughput, highly engineerable yeast cellular platform. Functional, properly folded GPCR yields ScFvs (which can easily be converted to IgG antibodies) or nanobodies that are more likely to function as therapeutics in the human organismal context.
- FIG. 1B shows use of an "antagonist selection strain” to find antagonists.
- FIG. 1C shows use of an "agonist selection strain” to find agonists.
- FIG. 1D shows direct functional screening yields therapeutic antibody candidates that would normally be missed in traditional screening, which could yield novel binding modes and functional modulation of GPCR targets.
- FIG. 2 shows method of reducing background/false positive in "halo assays”.
- 10 7 cells of the parental strain (left) and the current platform strain (NIY326, right) were plated on agar media.
- a filter paper disc was placed onto the plate and spotted with 3 m ⁇ of 1 mM alpha factor.
- a zone of no-growth in response to ligand (the desired phenotypic response) was observed in both, but in the parental strain (Left), suppressor mutants arise and grow into colonies in the presence of pheromone (colonies in halo region).
- In platform strain NI326 (Right) we have reduced the background rate to ⁇ l0 7 , as demonstrated in the clear halo zone and lack of background suppressor mutations that would act as false positives in an antagonist selection.
- FIG. 3 shows affinity molecule targeting vector structure and concept.
- affinity molecule downstream of a secretion signal and upstream of a linker and extracellular membrane-anchoring domain from GPI.
- the protein When expressed in cells, the protein is secreted into the extracellular space, and then the GPI domain is processed to leave a domain with a GPI that binds to the membrane, which tethers the affinity molecules to this cell and leaves it free to interact with the target GPCR on its extracellular face.
- FIGS. 4A and 4B show verification of affinity molecule expression/targeting vector.
- FIG. 4A shows that if an expressed anti-GFP nanobody properly folds and localizes, a GFP applied from the outside of the cell (after cell wall digestion) should label the cell membrane.
- FIG. 4B shows images of yeast expressing an anti-GFP nanobody using our targeting vector, after cell wall digestion and applying purified GFP protein indicate GFP binding at the membrane. No fluorescence was observed in control cells (data not shown) Left, brightfield; Right, GFP channel.
- FIGS. 5A and 5B show verification of the plasmid dependence of alpha factor resistant clones.
- FIG. 5A shows a schematic of the strategy.
- FIG. 5B shows an example of a "candidate" clone that exhibited alpha factor-resistant growth as analyzed by a halo assay, and then showed no resistance after forcing the plasmid to drop.
- FIG. 6 shows a workflow schematic.
- FIG. 7 shows the impact on growth rate of yeast cells by activation of the cannabinoid receptor type 2 (CB2 receptor) using VHH domain agonists displayed in the periplasmic space in various ways.
- CBD2 receptor cannabinoid receptor type 2
- Our innovation includes combining cell membrane-associated protein-to-yeast pheromone response coupling and expressing affinity molecules that act in cis in the same cell in a high-throughput platform. This enables direct and high-throughput functional selection of affinity molecules in the yeast periplasmic space.
- Antibodies and related affinity molecules are large molecules with complex folding patterns that must be maintained to retain binding activity. While unstructured, short peptides may be able to be localized to the yeast periplasmic space, it was not previously known that antibodies and related affinity molecules could be displayed in the yeast periplasmic space and retain binding activity.
- polypeptide and protein refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full length proteins and fragments thereof are encompassed by the definition.
- the terms also include post expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, and the like.
- a "polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions to the native sequence. These modifications may be deliberate, as through site directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
- antibody encompasses monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies.
- the term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293- 299; and U.S. Pat. No. 4,816,567); F(ab') 2 and F(ab) fragments; F v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al.
- the phrase "specifically (or selectively) binds" with reference to binding of an antibody to an antigen refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies.
- an antigen e.g., GPCR
- the specified antibodies bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample.
- Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen.
- antibodies raised to an antigen from specific species can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen.
- solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
- a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
- a protein is said to "interact” with another protein if it binds specifically (e.g., in a lock- and-key type mechanism), non-specifically or in some combination of specific and non-specific binding.
- a first protein "interacts preferentially” with a second protein if it binds (non- specifically and/or specifically) to the second protein with greater affinity and/or greater specificity than it binds to other proteins.
- affinity refers to the strength of binding and can be expressed quantitatively as a dissociation constant (3 ⁇ 4). It is to be understood that specific binding does not necessarily require interaction between specific amino acid residues and/or motifs of each protein.
- a first protein may interact preferentially with a second protein but, nonetheless, may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest).
- a weak, yet detectable, level e.g. 10% or less of the binding shown to the polypeptide of interest.
- weak binding, or background binding is readily discernible from the preferential interaction with the compound or polypeptide of interest, e.g., by use of appropriate controls.
- binding pair refers to first and second molecules that specifically bind to each other. "Specific binding" of the first member of the binding pair to the second member of the binding pair in a sample is evidenced by the binding of the first member to the second member, or vice versa, with greater affinity and specificity than to other components in the sample.
- the binding between the members of the binding pair is typically noncovalent. Examples include antigen-antibody, receptor-hormone, receptor-ligand, receptor-agonist, and receptor-antagonist binding pairs.
- ligand refers to a molecule that binds to another molecule, e.g., an antigen binding to an antibody, a hormone, agonist, or antagonist binding to a receptor, a neurotransmitter binding to an ion channel, or a substrate, inhibitor, or allosteric effector binding to an enzyme and includes natural and synthetic biomolecules, such as proteins, polypeptides, peptides, nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins, small molecules, natural and synthetic organic and inorganic materials, synthetic polymers, aptamers, and the like.
- polynucleotide generally refers to a nucleic acid molecule.
- a “polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic RNA and DNA sequences from viral (e.g. RNA and DNA viruses and retroviruses), prokaryotic DNA or eukaryotic (e.g., mammalian) DNA, and especially synthetic DNA sequences.
- the term also captures sequences that include any of the known base analogs of DNA and RNA, and includes modifications such as deletions, additions and substitutions (generally conservative in nature), to the native sequence. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts including polynucleotides encoding variant polypeptides for display.
- Modifications of polynucleotides may have any number of effects including, for example, facilitating expression of the polypeptide product in a host cell.
- a polynucleotide can encode a biologically active protein or polypeptide.
- a polynucleotide can include as little as 10 nucleotides, e.g., where the polynucleotide encodes an antigen or epitope.
- the polynucleotide encodes peptides of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or even more amino acids.
- variant refers to biologically active derivatives of the reference molecule that retain desired activity (e.g., efficient polypeptide display) as described herein.
- desired activity e.g., efficient polypeptide display
- variant and analog refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions and/or deletions relative to the native molecule, as long as the modifications do not destroy biological activity and which are "substantially homologous" to the reference molecule as defined below.
- amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned.
- the analogs will include the same number of amino acids but will include substitutions, as explained herein.
- mutant further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like.
- the term also includes molecules comprising one or more N-substituted glycine residues (a "peptoid") and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos.
- Analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
- amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
- Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
- the polypeptide of interest may include up to about 5-10 conservative or non conservative amino acid substitutions, or even up to about 15-25 conservative or non conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact.
- One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.
- Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature.
- the term "recombinant” as used with respect to a protein, polypeptide, or peptide means a polypeptide produced by expression of a recombinant polynucleotide.
- the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
- a "polynucleotide coding sequence” or a sequence that "encodes” a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mR A) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements").
- the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
- a transcription termination sequence may be located 3' to the coding sequence.
- control elements include, but are not limited to, transcription regulators, such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences; and translation regulators, such as sequences for optimization of initiation of translation, e.g., Shine-Dalgamo (ribosome binding site) sequences, Kozak sequences (i.e., sequences for the optimization of translation, located, for example, 5’ to the coding sequence), leader sequences (heterologous or native), translation initiation codon (e.g., ATG), and translation termination sequences.
- transcription regulators such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences
- translation regulators such as sequences for optimization of initiation of translation, e.g., Shine-Dalgamo (ribosome binding site) sequences, Kozak sequences (i.e., sequences for the optimization of translation, located, for example, 5’ to
- Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
- operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
- a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
- the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
- intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
- fragment is intended a molecule consisting of only a part of the intact full-length sequence and structure.
- the fragment can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the peptide.
- Active fragments of a particular protein or peptide will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity.
- substantially purified generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides.
- a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
- Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
- isolated is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type.
- isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
- Homology refers to the percent identity between two polynucleotide or two polypeptide molecules.
- Two nucleic acid, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules.
- substantially homologous also refers to sequences showing complete identity to the specified sequence.
- identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
- nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
- Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
- Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BEAST, used with default parameters.
- homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
- DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system.
- Expression cassette or "expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
- An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
- the expression cassette described herein may be contained within a plasmid or viral vector construct.
- the construct may also include, one or more selectable markers, a signal which allows the construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and an origin of replication (e.g., autonomously replicating sequence in yeast).
- a signal which allows the construct to exist as single stranded DNA e.g., a M13 origin of replication
- at least one multiple cloning site e.g., autonomously replicating sequence in yeast.
- transfection is used to refer to the uptake of foreign DNA by a cell.
- a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
- transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197.
- Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
- the term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
- a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
- target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
- vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
- expression vector e transfer vector
- the term includes cloning and expression vehicles, as well as plasmid and viral vectors.
- transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
- the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
- Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
- a "coding sequence” or a sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements").
- the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
- a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
- a transcription termination sequence may be located 3' to the coding sequence.
- control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
- label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
- fluorescer refers to a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range.
- labels include, but are not limited to radiolabels (e.g., H, I, S, C, or P), stable (non radioactive) heavy isotopes (e.g., 13 C or 15 N), phycoerythrin, fluorescein, 7-nitrobenzo-2-oxa-l,3- diazole (NBD), YPet, CyPet, Cascade blue, allophycocyanin, Alexa dyes (e.g., Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 594, Alexa 647, Alexa 660, Alexa 680, and Alexa 750), Atto dyes (e.g., Atto 488, Atto 532, Atto 550, Atto 565, Atto 590, Atto 610, Atto 620, Atto 635, Atto 647, Atto 655, and Atto 680), cyan
- the present invention is based on the development of methods for displaying
- recombinant proteins in the periplasmic space of yeast cells are linked to a cell membrane-spanning transmembrane domain, a cell-membrane associated protein domain that is on the external face of the yeast cell membrane, a protein that binds to the inner face of the yeast cell wall, or a periplasmic protein in order to display proteins in the yeast periplasmic space.
- Recombinant proteins can also be targeted to the periplasm by linking the recombinant protein to a secretion signal.
- a target protein of interest can be coexpressed in yeast such that it is localized to the plasma membrane or periplasmic space and accessible to displayed proteins.
- the inventors have used their method of yeast periplasmic display to screen for antibodies that bind to and modulate the function of human GPCRs (see Examples).
- Antibodies displayed in the periplasmic space of a yeast cell are in sufficient proximity to bind to a GPCR expressed in the cell membrane.
- the inventors have further developed a method for high-throughput screening of GPCRs for antagonists and agonists using periplasmic display by coupling human GPCRs to the yeast pheromone response pathway.
- yeast periplasmic display and methods of using it for high-throughput screening of protein libraries.
- the invention relates to the display of a protein in the periplasmic space of a yeast host cell.
- the yeast host cell comprises a protein for display in the yeast periplasmic space, a periplasm anchor protein linked to the protein to be displayed in the periplasmic space, and a target membrane protein of interest located in the periplasmic space of the yeast host cell.
- the yeast host cell can be used to determine if the protein to be displayed in the periplasmic space specifically binds to or affects the function of the target membrane protein of interest.
- the protein to be displayed in the periplasmic space of the yeast host cell can be prepared by linking a recombinant protein to a periplasm anchor protein that localizes the recombinant protein to the periplasmic space of a yeast cell.
- Linkage can be covalent or noncovalent.
- a recombinant protein may be linked covalently to a periplasm anchor protein in a fusion protein.
- a recombinant protein variant may form a complex with a periplasmic anchor protein, wherein the recombinant protein and the periplasmic anchor protein are linked noncovalently by molecular binding interactions in the complex.
- a recombinant protein variant and the periplasmic anchor protein are linked covalently by a non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- a protein to be displayed in the periplasmic space of the yeast host cell can also be prepared by linking the protein to be displayed to a secretion signal.
- the genus of the yeast host cell is selected from the group consisting of
- Saccharomyces Candida, Pichia, Kluyveromyces, and Yarrowia.
- the genus of the yeast host cells is Saccharomyces.
- the species of the yeast host cell is Saccharomyces cerevisiae.
- the invention relates to an antibody linked to a periplasm anchor protein.
- the antibody linked to a periplasm anchor protein further comprises an additional modification, moiety or interacting protein.
- the additional modification is a post-translational modification.
- the moiety is an affinity tag, epitope, label or the like.
- the antibody is localized to a yeast host cell periplasmic space. In some embodiments, when the antibody is produced in or introduced to a yeast host cell, the antibody is localized to a yeast host cell periplasmic space. In some embodiments, the antibody is linked to a periplasm anchor protein such that the antibody is localized to the periplasmic space.
- Linkage of the antibody to the periplasm anchor protein can be covalent or noncovalent.
- an antibody may be linked covalently to a periplasm anchor protein in a fusion protein.
- an antibody may form a complex with a periplasmic anchor protein, wherein the antibody and the periplasmic anchor protein are linked noncovalently by molecular binding interactions in the complex.
- an antibody and the periplasmic anchor protein are linked covalently by a non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- yeast host cells comprising an antibody as described herein.
- the genus of the yeast host cell is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the genus of the yeast host cells is Saccharomyces. In some embodiments, the species of the yeast host cell is Saccharomyces cerevisiae. [0109] In another aspect, the invention relates to methods of localizing an antibody to a yeast host cell periplasmic space comprising linking the antibody to a periplasm anchor protein such that the antibody is localized to the periplasmic space. In some embodiments, the antibody is linked to a periplasm anchor protein such that the antibody is localized to the periplasmic space.
- Linkage of the antibody to the periplasm anchor protein can be covalent or noncovalent.
- an antibody may be linked covalently to a periplasm anchor protein in a fusion protein.
- an antibody may form a complex with a periplasmic anchor protein, wherein the antibody and the periplasmic anchor protein are linked noncovalently by molecular binding interactions in the complex.
- an antibody and the periplasmic anchor protein are linked covalently by a non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- the genus of the yeast host cell is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the genus of the yeast host cells is Saccharomyces. In some embodiments, the species of the yeast host cell is Saccharomyces cerevisiae.
- the invention relates to methods of high-throughput screening of protein libraries for specific binding or functional characteristics by displaying proteins in the periplasmic space of yeast.
- a yeast periplasmic display library can be prepared by linking recombinant proteins to a periplasm anchor protein that localizes recombinant proteins to the periplasmic space of a yeast cell. Linkage can be covalent or noncovalent.
- a recombinant protein may be linked covalently to a periplasm anchor protein in a fusion protein.
- a recombinant protein variant may form a complex with a periplasmic anchor protein, wherein the recombinant protein and the periplasmic anchor protein are linked noncovalently by molecular binding interactions in the complex.
- a recombinant protein variant and the periplasmic anchor protein are linked covalently by a non-peptidic bond in a complex.
- the non-peptidic bond is a disulfide bond.
- a yeast periplasmic display library can also be prepared by linking recombinant proteins to secretion signals.
- the genus of the yeast host cell is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia.
- the genus of the yeast host cells is Saccharomyces.
- the species of the yeast host cell is Saccharomyces cerevisiae.
- a periplasm anchor protein comprises a signal sequence that directs transport of the periplasm anchor protein to the yeast host cell periplasm, plasma membrane, or cell wall such that a linked recombinant protein is displayed in the periplasm.
- the periplasm anchor protein may comprise a signal sequence that directs transport of the periplasm anchor protein and the linked recombinant protein to the yeast host cell periplasm.
- the periplasm anchor protein is sufficiently large that the periplasm anchor protein and the linked recombinant protein are retained in the periplasm.
- the periplasm anchor protein may be a component of a periplasmic protein complex that is sufficiently large that formation of the complex in the periplasm results in retention in the periplasm.
- the periplasm anchor protein comprises a membrane-spanning transmembrane domain or a membrane-associated protein domain that localizes to an external face of the cell membrane such that the linked recombinant protein is projected into the periplasm.
- a glycosylphosphatidylinositol (GPI)-anchoring domain that localizes to the plasma membrane can be used for this purpose.
- the GPI-plasma membrane anchoring domain is a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS 1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin
- the periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the displayed protein variant is projected into the periplasm.
- the periplasm anchor protein and the recombinant protein are covalently linked in a fusion protein variant and the periplasm anchor protein comprises a signal sequence that directs transport of the fusion protein to the yeast host cell periplasm, plasma membrane, or cell wall such that the fused protein variant is displayed in the periplasm.
- the periplasm anchor protein may comprise a signal sequence that directs transport of the fusion protein to the yeast host cell periplasm.
- the periplasm anchor protein is sufficiently large that the fusion protein is retained in the periplasm.
- the periplasm anchor protein may be a component of a periplasmic protein complex that is sufficiently large that formation of the complex in the periplasm results in retention of the fusion protein in the periplasm.
- the periplasm anchor protein comprises a membrane-spanning transmembrane domain or a membrane-associated protein domain that localizes to an external face of the cell membrane such that the fused protein variant is projected into the periplasm.
- a glycosylphosphatidylinositol (GPI)- anchoring domain that localizes to the plasma membrane can be used for this purpose.
- the GPI-plasma membrane anchoring domain is a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS 1, YPS2, YPS3, YPS4, YPS5, YPS6, or
- the periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the displayed protein variant is projected into the periplasm.
- the periplasm anchor protein is a fragment of a full-length protein that retains the ability to be localized to the periplasm.
- Any type of protein may be displayed in the periplasmic space of a yeast cell, including, but not limited to antibodies, antibody mimetics, aptamers, antigens, enzymes, receptors, transporters, ion channels, hormones, substrates, agonists, antagonists, or ligands.
- the yeast periplasmic display library presents a plurality of such proteins, which can be screened for binding and/or biological activity in the presence of a target molecule of interest. If located extracellularly, the target molecule must be able to penetrate the yeast cell wall to reach the displayed proteins for screening.
- a target protein of interest is coexpressed with a displayed protein variant in a yeast cell at a location accessible to the displayed protein variant (e.g., in close enough proximity for the displayed protein variant to bind to and/or modulate the activity of the target protein of interest).
- the target protein of interest may be localized to the yeast host cell plasma membrane or periplasm near where the protein variant is displayed.
- this method is applicable to receptors, ion channels, transporters, and other membrane proteins which localize to the plasma membrane.
- yeast periplasmic display can be used to present proteins to a target membrane protein in an environment substantially similar to its native environment.
- Any polypeptides included in a fusion construct, including the periplasm anchor protein and displayed protein variant may be connected directly to each other by peptide bonds or may be separated by intervening amino acid sequences or linkers.
- Linker amino acid sequences will typically be short, e.g., 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1).
- Linkers may include restriction sites, which aid cloning and manipulation.
- Other suitable linker amino acid sequences will be apparent to those skilled in the art. (See e.g., Argos (1990) J. Mol. Biol.
- a tag may be included in fusion constructs.
- Tags that can be used in the practice of the invention include, but are not limited to a His-tag, a Strep-tag, a TAP -tag, an S- tag, an SBP-tag, an Arg-tag, a calmodulin-binding peptide tag, a cellulose-binding domain tag, a DsbA tag, a c-myc tag, a glutathione S-transferase tag, a FLAG tag, a HAT-tag, a maltose binding protein tag, a NusA tag, and a thioredoxin tag.
- Polynucleotides encoding periplasm-anchored protein variants and target proteins of interest can be produced in any number of ways, all of which are well known in the art. For example, polynucleotides can be generated using recombinant techniques, well known in the art. One of skill in the art can readily determining nucleotide sequences that encode the desired proteins using standard methodology and the teachings herein.
- Oligonucleotide probes can be devised based on known gene sequences and used to probe genomic or cDNA libraries. The polynucleotides with desired sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate a gene at desired portions of the full-length sequence. Similarly, polynucleotides with sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce desired protein variants. See, e.g., Sambrook et ah, supra, for a description of techniques used to obtain and isolate DNA.
- sequences encoding protein variants can also be produced synthetically, for example, based on known sequences.
- the nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired.
- the complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259:6311; Stemmer et al. (1995) Gene 164:49-53.
- Recombinant techniques are readily used to clone sequences encoding protein variants (e.g., antibodies) useful in the claimed invention that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid.
- a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes.
- the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex.
- the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located.
- Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
- the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409.
- the diversity of a display library will depend on the method of mutagenesis that is used.
- Cassette mutagenesis can be used to quickly generate a large number of mutations by insertion of mutagenic cassettes into a nucleic acid (see, e.g., Worrall (1994) Methods Mol. Biol. 30: 199-210, Kegler-Ebo et al. (1994) Nucleic Acids Research. 22 (9): 1593-1599).
- random mutagenesis can also be used to generate large numbers of variants for a display library.
- Suitable methods of random mutagenesis include, but are not limited to, error-prone PCR, rolling circle error-prone PCR, chemical mutagenesis, mutagenesis in a mutator strain with deficient DNA repair pathways, insertion mutagenesis using a transposon system, or DNA shuffling (see, e.g., McCullum et al. (2010) Methods Mol. Biol. 634: 103-109, Fujii et al. (2014) Methods Mol. Biol. 1179:23-29, Muteeb (2010) Methods Mol. Biol. 634:411-419, Bose (2016) Methods Mol. Biol . 1373 : 111 - 115 , Labrou (2010) Curr Protein Pept Sci.
- a DNA library is constructed containing at least 10 6 , preferably at least 10 8 , and more preferably at least 10 10 variants with unique sequences, using methods known in the art.
- antibody libraries are constructed for yeast periplasmic display by cloning natural antibodies from B lymphocytes obtained from blood donors. Nucleic acids encoding antibody light and heavy chains or fragments thereof containing variable domain complementarity-determining regions (e.g., Fab) can be amplified by PCR and cloned into vectors. ScFv antibodies can be generated by cloning into a vector that connects the light and heavy chains via a linker in one open reading frame.
- the blood donor can be of any species.
- human blood donors are used for generation of a library of human antibodies.
- camelid blood donors are used for generation of a library of camelid antibodies.
- Camelid antibodies may be derived, for example, from Dromedary camels, bactrian camels, llamas and alpacas. Such camelids produce a unique type of antibody that lacks a light chain.
- These heavy-chain antibodies (HCAbs) or variable domain fragments thereof (e.g., single-domain antibodies or nanobodies) can be used to construct an antibody library (see, e.g., Vincke et al. (2012) Methods Mol. Biol. 911: 15-26, Krah et al. (2016) Immunopharmacol.
- a "vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell.
- vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- the term “vector” includes an autonomously replicating plasmid or a virus.
- An expression construct can be replicated in a living cell, or it can be made synthetically.
- the terms "expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.
- the nucleic acid encoding a polynucleotide of interest is under transcriptional control of a promoter.
- a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
- the term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III. Enhancer elements may be used in association with the promoter to increase expression levels of the constructs.
- an expression vector comprises a promoter "operably linked" to a polynucleotide encoding a fusion protein or target protein of interest.
- operably linked or "under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the fusion protein or target protein of interest.
- transcription terminator/polyadenylation signals will also be present in the expression construct.
- sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al, supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Patent No. 5,122,458).
- 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same.
- Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
- IRES internal ribosome entry site
- the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997 22 150-161.
- a multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of
- picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125- 15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al, J Biol. Chem.
- EMCV encephalomyocarditis virus
- UTR the polio leader sequence
- the hepatitis A virus leader the hepatitis C virus IRES
- human rhinovirus type 2 IRES Dobrikova et al., Proc. Nat
- IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al ,Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-l IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(l7):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A.
- IRES insulin-like growth factor 2
- Clontech Mountain View, CA
- Invivogen San Diego, CA
- Addgene Cambridge, MA
- GeneCopoeia Rockville, MD
- An IRES sequence may be included in a vector, for example, to express a fusion protein comprising a periplasm anchor protein fused to a protein variant for display in combination with a target protein of interest from an expression cassette.
- a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products from a single vector.
- 2A linker peptides are inserted between the coding sequences in the multicistronic construct.
- the 2A peptide which is self cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels.
- 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus- 1. See, e.g., Kim et al.
- the expression construct comprises a plasmid suitable for transforming a yeast cell.
- Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, MET15, ura4+, leul+, ade6+), antibiotic selection markers (e.g., aphAl or ble), fluorescent markers (e.g., mCherry, green fluorescent protein), bioluminescent markers (e.g., luciferase), or other markers for selection of transformed yeast cells.
- the yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E. coli) and yeast cells.
- yeast plasmids A number of different types are available including yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.
- Yip yeast integrating plasmids
- ARS autonomously replicating sequence
- YCp yeast centromere plasmids
- CEN yeast episomal plasmids
- yeast episomal plasmids YEp
- regulatory sequences may also be desirable, which allow for regulation of expression of the protein sequences relative to the growth of the host cell.
- Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
- a pheromone-inducible promoter such as a PRM1 or FUS2 promoter can be used to make transcription dependent on activation of the pheromone signaling pathway.
- the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
- the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
- Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site- directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et ah, supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.
- recombinant polynucleotides encoding protein variants are cloned into a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a polynucleotide encoding a protein variant after the polynucleotide encoding the signal peptide; c) a polynucleotide encoding a glycophosphatidylinositol (GPI) plasma membrane anchoring domain, positioned such that the vector is capable of producing a fusion protein comprising the signal peptide and the protein variant fused to the GPI plasma membrane anchoring domain; and d) a promoter operably linked to sequences encoding the fusion protein.
- a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a clo
- the GPI plasma membrane anchoring domain is a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- the periplasm-targeting expression vector may further comprise a polynucleotide encoding a linker positioned in between the cloning site and the polynucleotide encoding the GPI plasma membrane anchoring domain.
- the cloning site may comprise one or more restriction sites.
- the periplasm-targeting expression vector may further comprise a selectable marker.
- an affinity tag refers to a biomolecule, such as a polypeptide segment, that can be attached to a second biomolecule to provide for purification or detection of the second biomolecule or provide sites for attachment of the second biomolecule to a substrate.
- affinity tags include a poly-histidine tract, protein A (Nilsson et al. (1985) EMBO J. 4: 1075; Nilsson et al. (1991) Methods Enzymol.
- a "label” is a molecule or atom which can be conjugated to a biomolecule to render the biomolecule or a form of the biomolecule, such as a conjugate, detectable or measurable.
- labels include fluorescent agents, bioluminescent proteins, photoactive agents, radioisotopes, paramagnetic ions, chelators, and the like.
- yeast hosts are known in the art, including but not limited to, Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus , Saccharomyces eubayanus,
- Saccharomyces exiguus Saccharomyces florentinus
- Saccharomyces fragilis Saccharomyces kluyveri
- Saccharomyces kudriavzevii Saccharomyces martiniae
- Saccharomyces mikatae Saccharomyces monacensis
- Saccharomyces norbensis Saccharomyces paradoxus
- Saccharomyces pastorianus Saccharomyces spencerorum, Saccharomyces turicensis,
- Candida tolerans Candida tropicalis, Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candida subhashii, Candida viswanathii, Candida utilis, Candida ubatubensis, Candida zemplinina, Pichia farinosa, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, Pichia subpelliculosa, Kluyveromyces aestuarii, Kluyveromyces marinus, Kluyveromyces bacillisporus , Kluyveromyces blattae, Kluyveromyces dobzhanskii,
- Kluyveromyces hubeiensis Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Kluyveromyces nonfermentans, Kluyveromyces piceae, Kluyveromyces sinensis, Kluyveromyces thermotolerans, Kluyveromyces waltii, Kluyveromyces wickerhamii,
- the yeast is a Saccharomyces species. In some embodiments, the yeast is Saccharomyces cerevisiae.
- the expression construct In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a yeast cell. This delivery may be accomplished in vitro using laboratory procedures for transforming yeast cells well-known in the art, such as spheroplast transformation, alkaline ion treatment (e.g., Cs + or Li + ), electroporation, trans-kingdom conjugation, electroporation, and biolistic and glass bead methods (see, e.g., Kawai et al. (2010) Bioeng. Bugs. l(6):395-403, Gietz et al. (1995) Yeast 11(4):355-360, Gietz et al. (2007) Nat. Protoc.
- alkaline ion treatment e.g., Cs + or Li +
- electroporation e.g., electroporation, trans-kingdom conjugation, electroporation, and biolistic and glass bead methods
- biolistic and glass bead methods see, e
- the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
- the nucleic acid encoding the gene may be stably integrated into the genome of the cell via homologous recombination. This integration may be in the cognate location and orientation (gene replacement), within a gene (gene disruption), or in a random, non-specific location (gene augmentation). Integration of a construct at a target locus that disrupts a gene may be acceptable as long as the gene disruption does not interfere with cell growth or screening of the yeast periplasmic display library (e.g., avoid disruption of pheromone response if used in screening).
- the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
- Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
- the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
- a naked DNA expression construct may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce yeast cell walls and membranes and enter cells without killing them (Armaleo et al. (1990) Curr. Genet. 17(2):97-103).
- Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Y ang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572).
- the microprojectiles may consist of biologically inert substances, such as tungsten or gold beads.
- a collection of linear DNA molecules encoding protein variants are generated. Rather than cloning the linear DNA molecules into a vector prior to
- the yeast cells are transformed with an empty vector together with the collection of linear DNA molecules encoding the protein variants, which subsequently integrate into the vector in vivo, e.g., by homologous recombination in the yeast host cells.
- a kit may include a yeast periplasmic display library, as described herein, or agents for preparing a yeast periplasmic display library, such as suitable vectors for cloning nucleic acids encoding protein variants for production of protein variants linked to a periplasm anchor protein, yeast cells, transfection agents, media suitable for growing yeast cells, agents for positive and negative selection of cells, and other reagents that are required. Instructions (e.g., written, CD- ROM, DVD, Blu-ray, flash drive, digital download, etc.) for production and/or screening of a yeast periplasmic display library as described herein usually will be included in the kit.
- agents for preparing a yeast periplasmic display library such as suitable vectors for cloning nucleic acids encoding protein variants for production of protein variants linked to a periplasm anchor protein, yeast cells, transfection agents, media suitable for growing yeast cells, agents for positive and negative selection of cells, and other reagents that are required.
- Instructions e.g.
- a kit may include a yeast periplasmic display library, as described herein, or agents for preparing a yeast periplasmic display library, such as suitable vectors for cloning nucleic acids encoding protein variants for production of fusions with a periplasm anchor protein, yeast cells, transfection agents, media suitable for growing yeast cells, agents for positive and negative selection of cells, and other reagents that are required. Instructions (e.g., written, CD-ROM, DVD, Blu-ray, flash drive, digital download, etc.) for production and/or screening of a yeast periplasmic display library as described herein usually will be included in the kit.
- agents for preparing a yeast periplasmic display library such as suitable vectors for cloning nucleic acids encoding protein variants for production of fusions with a periplasm anchor protein, yeast cells, transfection agents, media suitable for growing yeast cells, agents for positive and negative selection of cells, and other reagents that are required.
- Instructions e.g., written,
- the kit comprises a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a polynucleotide encoding a protein variant (e.g., antibody) after the polynucleotide encoding the signal peptide; c) a polynucleotide encoding a glycophosphatidylinositol (GPI) plasma membrane anchoring domain, positioned such that the vector is capable of producing a fusion protein comprising the signal peptide and the protein variant fused to the GPI plasma membrane anchoring domain; and d) a promoter operably linked to sequences encoding the fusion protein.
- a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a poly
- the signal peptide comprises a prepro-alpha-factor signal sequence.
- the GPI plasma membrane anchoring domain is a yapsin GPI plasma membrane anchoring domain such as, but not limited to, a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- the periplasm-targeting expression vector further comprises a polynucleotide encoding a linker, wherein said polynucleotide encoding the linker is positioned in between the cloning site and the polynucleotide encoding the GPI plasma membrane anchoring domain.
- the linker may further comprise a tag.
- the periplasm-targeting expression vector further comprises a selectable marker.
- the cloning site comprises one or more restriction sites.
- the kit includes a yeast periplasmic display library comprising a plurality of yeast host cells, each yeast host cell comprising: a) a fusion protein comprising a periplasm anchor protein fused to a protein variant (e.g., antibody) for display in the yeast host cell periplasmic space, wherein the displayed protein variant is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of protein variants; b) a target G- protein coupled receptor (GPCR) of interest, wherein the target GPCR of interest is located in the yeast host cell plasma membrane and accessible to the protein variant displayed in the yeast host cell periplasmic space; and c) an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- a yeast periplasmic display library comprising a plurality of yeast host cells, each yeast host cell comprising: a) a fusion protein comprising a peri
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C- terminal domain of an exogenous Ga subunit.
- the yeast Ga subunit may belong to a Gai, Gaq, Gas, or Gao family G protein.
- the chimeric Ga subunit at least five C-terminal residues of a yeast Ga subunit may be replaced with corresponding C-terminal residues of a mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by a mammalian GPCR.
- the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR.
- the chimeric Ga subunit comprises at least 41 N-terminal residues of the yeast Ga subunit.
- the mammalian GPCR is a mouse GPCR.
- the mammalian GPCR is a human GPCR selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR, and AT1R.
- the kit includes a yeast periplasmic display library comprising: a plurality of yeast host cells, each yeast host cell comprising a fusion protein comprising a periplasm anchor protein fused to a protein variant for display, wherein the periplasm anchor protein is sufficiently large that the fusion protein is retained in the periplasm, and the displayed protein variant is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of protein variants.
- the kit includes a yeast periplasmic display library comprising: a plurality of yeast host cells, each yeast host cell comprising a fusion protein comprising a protein variant for display linked to a target membrane protein of interest, wherein the displayed protein variant is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of protein variants with the target membrane protein of interest.
- the present invention may be broadly applied in screening for proteins that perform useful or desired functions including binding, catalysis, assembly, transport, and the like.
- yeast periplasmic display can be used in identifying agonists and antagonists for receptors, engineering therapeutic proteins and antibodies, identifying protein-protein interactions, and epitope mapping.
- Yeast periplasmic display is particularly well-suited for screening a protein library for candidates that bind to and/or modulate the function of a target protein that is a membrane protein, such as a receptor, an ion channel, or a transporter. Localization of a protein to the membrane makes it accessible to the displayed protein variants in the periplasmic space (e.g., in close enough proximity for a displayed protein variant to bind to the target protein of interest).
- a membrane protein such as a receptor, an ion channel, or a transporter.
- the yeast periplasmic display library may be screened for an agonist of the target protein of interest by culturing at least a subset of the yeast host cells of the yeast periplasmic display library in a media, wherein growth of a yeast host cell in the media indicates that the protein variant displayed in the yeast host cell is an agonist of the target protein of interest.
- activation of the target protein of interest decreases growth of the yeast host cells.
- the yeast periplasmic display library may be screened for an antagonist of the target protein of interest by culturing at least a subset of the yeast host cells of the yeast periplasmic display library in a media, wherein growth of a yeast host cell in the media indicates that the protein variant displayed in the yeast host cell is an antagonist of the target protein of interest.
- each yeast host cell further comprises a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target protein of interest is activated to allow detection of increases or decreases in activity of the target protein of interest upon binding of the displayed protein variant to the target protein of interest.
- the reporter gene may be a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), antibiotic resistance marker (e.g., confers resistance to an antibiotic such as geneticin (e.g., aphAl), zeocin (e.g., ble), hygromycin B, nourseothricin, or bialaphos), fluorescent marker (e.g., a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein), bioluminescent marker (e.g., luciferase and aequorin), or counter-selectable marker (e g., CAN1, URA3, MET15, TRP1, and TK).
- antibiotic resistance marker e.g., confers resistance to an antibiotic such as geneticin (e.g., aphAl), zeocin (e.g., ble),
- positive selection i.e., selection for the activation of expression of the reporter gene
- expression of a nutritional marker can be detected, for example, by growth of the yeast host cells in a nutrient-deficient selection media.
- Expression of an antibiotic resistance marker can be detected, for example, by growth of the yeast host cells in a selection media comprising an antibiotic.
- Expression of a fluorescent marker can be detected, for example, by fluorescence emitted by the yeast host cells.
- Expression of a bioluminescent marker can be detected, for example, by bioluminescence emitted by the yeast host cells.
- counterselection i.e., growth-based selection for the loss of expression of the reporter gene
- counterselection i.e., growth-based selection for the loss of expression of the reporter gene
- a counter-selectable marker may kill cells by inducing apoptosis, converting a nontoxic drug to a toxic compound, or transporting a toxic molecule into a cell.
- Counterselection can be performed by culturing the yeast host cells in a media comprising an agent that selectively kills cells expressing the counter-selectable marker.
- Exemplary counter selectable markers include CAN1 (counterselection with canavanine), URA3 (counterselection with 5-fluoro-orotic acid (5-FOA)), MET15 (counterselection with methylmercury), TRP1 (counterselection with 5-fluoroanthranilic acid (5-FAA)), and human Herpes virus thymidine kinase TK (counterselection with floxuridine (FUDR)).
- CAN1 counterselection with canavanine
- URA3 counterselection with 5-fluoro-orotic acid
- MET15 counterselection with methylmercury
- TRP1 counterselection with 5-fluoroanthranilic acid
- FUDR floxuridine
- a yeast periplasmic display library may be used for screening for antibodies that bind to and modulate the function of a GPCR.
- a GPCR in the yeast host cell is replaced with a mammalian GPCR, e.g., human GPCR.
- the yeast host cell expresses a mammalian GPCR, e.g., human GPCR, and the endogenous yeast GPCR.
- antagonists and agonists are identified using a reporter system that couples the response of a GPCR to binding of a displayed antibody to levels of yeast pheromone secretion (see Examples).
- the yeast host cell can be genetically modified to express an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- the yeast Ga subunit may belong to a Gai, Gaq, Gas, or Gao family G protein.
- the chimeric Ga subunit at least five C- terminal residues of a yeast Ga subunit may be replaced with corresponding C-terminal residues of a mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by a mammalian GPCR. In some embodiments, at least 20 C-terminal residues of the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR. In another embodiment, the chimeric Ga subunit comprises at least 41 N-terminal residues of the yeast Ga subunit.
- Exemplary mammalian Ga subunits include G alpha-S, G alpha-I, G alpha-O, G alpha- T, G alpha-Z, G alpha-Q, G alpha-l 1, G alpha-l2, G alpha-l3, and transducin.
- a ⁇ mat strain may comprise, for example, a deleted or inactivated MATa locus or a deleted or inactivated MATa locus.
- the yeast host cell may further comprise a modified CLN3 protein comprising a C-terminal truncation that increases abundance of CLN3 in the yeast host cell compared to a wild-type CLN3 protein.
- the modified CLN3 protein may retain at least N-terminal amino acids 1-387 or 1-408 of the wild- type CLN3 protein, or any number of N-terminal amino acids within these ranges, such as 1-388, 1-389, 1-390, 1-391, 1-392, 1-393, 1-394, 1-395, 1-396, 1-397, 1-398, 1-399, 1-400, 1-401, 1- 402, 1-403, 1-404, 1-405, 1-406, 1-407, or 1-408, wherein the C-terminal truncation comprises a deletion of all or some of the remaining residues of the wild-type CLN3 protein.
- the yeast host cells used to prepare a periplasmic display library may be haploid or diploid. Exemplary yeast strains designed for use in antagonist and agonist selection are described in Examples 2 and 5, respectively.
- the yeast host cell is a FAR1 strain, wherein inhibition of the pheromone response by an antibody acting as an antagonist that binds to an inhibits the GPCR in the yeast host cell results in cessation of cell cycle arrest and growth of the yeast host cell.
- the yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds to and activates the GPCR in the yeast host cell results in increased expression of the reporter gene.
- Any type of GPCR from any species may be screened using periplasmic display as described herein.
- the target GPCR of interest is a mammalian GPCR (e.g., from human or nonhuman primate, rodent, laboratory animal, livestock).
- the mammalian GPCR may be a human GPCR (e g., CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR, and AT1R).
- the target GPCR of interest may have constitutive ligand-independent activity.
- a ligand may be added to activate the target GPCR of interest during screening for agonists or antagonists.
- the yeast host cell expresses the target GPCR of interest, e.g., human GPCR of interest, and the endogenous yeast GPCR.
- the protein variants are antibodies. Any type of antibody may be screened using yeast periplasmic display by the methods described herein, including monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example,
- the protein variants are aptamers.
- Aptamers may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection.
- Aptamers For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan,
- the protein variants are antibody mimetics. Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (11):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (l5):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5): 1058-1068), alphabodies (Desmet et al. (2014) Nature
- directed evolution with multiple rounds of mutagenesis and screening by yeast periplasmic display may be performed to enrich libraries for protein variants (e.g., antibodies) that bind with high affinity to a target protein of interest.
- Directed evolution may be particularly useful for improving the binding characteristics of candidates with desired functional activities but weak binding affinities.
- a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises:
- periplasm anchor protein b) a periplasm anchor protein, wherein the periplasm anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space;
- a target membrane protein of interest wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- periplasm anchor protein comprises a membrane -spanning transmembrane domain or a membrane associated protein domain that projects the antibody into the periplasm.
- GPI glycosylphosphatidylinositol
- yapsin GPI plasma membrane anchoring domain is a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the antibody is projected into the periplasm.
- periplasm anchor protein is a protein that binds to an inner face of the cell wall that projects the fusion protein into the periplasm.
- a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises:
- an antibody for display in the yeast host cell periplasmic space wherein the displayed antibody is different in each yeast host cell such that the plurality of yeast host cells displays a plurality of antibodies, wherein the antibody is linked to a signal sequence that directs transport of the antibody to the yeast host cell periplasm, plasma membrane or cell wall, such that the antibody is displayed in the yeast host cell periplasmic space;
- a target membrane protein of interest wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- yeast periplasmic display library of embodiment 15, wherein the nutritional marker is selected from the group consisting of HIS3, HIS7, ARG6, LEU2, URA3, and TRP1.
- GPCR G-protein coupled receptor
- Ga G protein alpha
- the yeast periplasmic display library of embodiment 35 wherein the human GPCR is selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR, and AT1R.
- yeast periplasmic display library of any one of embodiments 24-36 wherein the yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of the GPCR target membrane protein of interest.
- yeast periplasmic display library of any one of embodiments 1-40, wherein the yeast host cell is a ⁇ far 1. ⁇ sst2. ⁇ ste 14. ⁇ stc3. or ⁇ mat strain.
- yeast periplasmic display library of embodiment 42 wherein the yeast host cell is a ⁇ mat strain comprising a deleted or inactivated MATa locus or a deleted or inactivated MATa locus.
- yeast host cells e) culturing the plurality of yeast host cells under conditions that permit expression of the antibodies, the periplasm anchor protein and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest localizes to the plasma membrane, such that the yeast periplasmic display library of embodiment 1 is produced.
- each recombinant polynucleotide encodes a different fusion protein comprising the periplasm anchor protein linked to a different antibody for display;
- yeast periplasmic display library of embodiment 3 is produced.
- the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- yeast Ga subunit belongs to a Gai, Gaq, Gas, or Gao family G protein.
- yeast host cell is a FAR1 strain for selection of antibody antagonists of the target GPCR of interest.
- yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of the GPCR.
- a method of screening the yeast periplasmic display library of embodiment 14 for an antibody that modulates activity of the target membrane protein of interest comprising culturing at least a subset of the yeast host cells of the yeast periplasmic display library of embodiment 14 in a selection media; and detecting expression of the reporter gene, wherein increased expression of the reporter gene indicates that the antibody increases activity of target membrane protein of interest and decreased expression of the reporter gene indicates that the antibody decreases activity of the target membrane protein of interest.
- reporter gene is a nutritional marker, antibiotic resistance marker, fluorescent marker, biolumine scent marker, or a counter-selectable marker.
- nourseothricin nourseothricin, and bialaphos.
- the fluorescent marker is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein.
- a method of screening the yeast periplasmic display library of embodiment 27 for an antibody that modulates the activity of the target GPCR of interest comprising culturing at least a subset of the yeast host cells of the yeast periplasmic display library of embodiment 27 in a media, wherein detection of activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell not having an antibody displayed in the periplasmic space indicates that the displayed antibody in said at least one yeast host cell binds to and modulates the activity of the GPCR.
- yeast host cell is a FAR1 strain
- inhibition of the pheromone response by an antibody acting as an antagonist that binds to an inhibits the GPCR in the yeast host cell results in cessation of cell cycle arrest and growth of the yeast host cell.
- yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds to and activates the GPCR in the yeast host cell results in increased expression of the reporter gene.
- reporter gene is a nutritional marker, antibiotic resistance marker, fluorescent marker, biolumine scent marker, or a counter-selectable marker.
- a yeast host cell comprising:
- periplasm anchor protein b) a periplasm anchor protein, wherein the periplasm anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space;
- a target membrane protein of interest wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- the antibody and the periplasm anchor protein are noncovalently linked together by molecular binding interactions in a complex or are linked by a covalent non-peptidic bond in a complex.
- yeast host cell of embodiment 86 wherein the antibody and the periplasm anchor protein are covalently linked together in a fusion protein.
- periplasm anchor protein further comprises a signal sequence that directs transport of the periplasm anchor protein to the yeast host cell periplasm, plasma membrane, or cell wall such that the antibody is displayed in the periplasm.
- periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the antibody is projected into the periplasm.
- yeast host cell of any one of embodiments 86-92, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.
- yeast host cell of embodiment 93, wherein the receptor is a G-protein coupled receptor (GPCR).
- GPCR G-protein coupled receptor
- yeast host cell of any one of embodiments 86-94 further comprising introducing into the yeast host cell a recombinant polynucleotide encoding an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the yeast host cell of embodiment 95 wherein the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C- terminal domain of an exogenous Ga subunit.
- Ga G protein alpha
- yeast host cell of embodiment 96 wherein the yeast Ga subunit belongs to a Gai, Gaq, Gas, or Gao family G protein.
- yeast host cell of embodiment 99 wherein at least 20 C-terminal residues of the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR.
- yeast host cell of any one of embodiments 86-101 wherein the yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of the GPCR.
- yeast host cell of embodiment 103 wherein the genus of the yeast host cells is Saccharomyces.
- yeast host cell of embodiment 104 wherein the species of the Saccharomyces is Saccharomyces cerevisiae.
- periplasm anchor protein further comprises a signal sequence that directs transport of the periplasm anchor protein to the yeast host cell periplasm, plasma membrane, or cell wall such that the antibody is displayed in the periplasm.
- periplasm anchor protein comprises a membrane-spanning transmembrane domain or a membrane associated protein domain that projects the antibody into the periplasm.
- the membrane associated protein domain is a glycosylphosphatidylinositol (GPI)-plasma membrane anchoring domain.
- periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the antibody is projected into the periplasm.
- a yeast host cell comprising the antibody of any one of embodiments 106-119.
- a method of localizing an antibody to a yeast host cell periplasmic space comprising linking the antibody to a periplasm anchor protein such that the antibody is localized to the periplasmic space.
- periplasm anchor protein further comprises a signal sequence that directs transport of the periplasm anchor protein to the yeast host cell periplasm, plasma membrane, or cell wall such that the antibody is displayed in the periplasm.
- periplasm anchor protein comprises a membrane-spanning transmembrane domain or a membrane associated protein domain that projects the antibody into the periplasm.
- the membrane associated protein domain is a glycosylphosphatidylinositol (GPI)-plasma membrane anchoring domain.
- periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the antibody is projected into the periplasm.
- a yeast periplasmic display library comprising a plurality of yeast host cells, wherein each yeast host cell comprises:
- periplasm anchor protein b) a periplasm anchor protein, wherein the periplasm anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space;
- a target membrane protein of interest wherein the membrane protein of interest is located in the yeast host cell plasma membrane and accessible to the antibody displayed in the yeast host cell periplasmic space.
- yeast periplasmic display library of embodiment 133 wherein the antibody and the periplasm anchor protein are covalently linked together in a fusion protein.
- the yeast periplasmic display library of embodiment 133 wherein the antibody and the periplasm anchor protein are noncovalently linked together by molecular binding interactions.
- the yeast periplasmic display library of embodiment 133 further comprising a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target membrane protein of interest is activated to allow detection of increases or decreases in activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest.
- the yeast periplasmic display library of embodiment 136 wherein the reporter gene is a nutritional marker, antibiotic resistance marker, fluorescent marker, bioluminescent marker, or counter-selectable marker.
- the yeast periplasmic display library of embodiment 137 wherein the nutritional marker is selected from the group consisting of HIS3, HIS7, ARG6, LEU2, URA3, and TRP1.
- the yeast periplasmic display library of embodiment 137 wherein the antibiotic resistance marker confers resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.
- the yeast periplasmic display library of embodiment 137 wherein the fluorescent marker is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein.
- yeast periplasmic display library of embodiment 137 wherein the bioluminescent marker is luciferase or aequorin.
- the yeast periplasmic display library of embodiment 137 wherein the counter-selectable marker is selected from the group consisting of CAN1, URA3, MET15, TRP1, and TK.
- the yeast periplasmic display library of embodiment 136 wherein the reporter gene is a counter-selectable marker such that said decreases in activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest are detectable by growth of the yeast host cells on a negative selection media.
- the yeast periplasmic display library of embodiment 133 wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.
- GPCR G- protein coupled receptor
- the yeast periplasmic display library of embodiment 146 wherein the GPCR is an exogenous GPCR.
- the yeast periplasmic display library of embodiment 147 further comprising an engineered Ga subunit capable of being activated by the exogenous GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the yeast periplasmic display library of embodiment 148 wherein the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- Ga G protein alpha
- yeast periplasmic display library of embodiment 149 wherein the yeast Ga subunit belongs to a Gai, Gaq, Gas, or Gao family G protein.
- the yeast periplasmic display library of embodiment 151 wherein at least five C-terminal residues of the yeast Ga subunit are replaced with corresponding C-terminal residues of a mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR.
- yeast periplasmic display library of embodiment 152 wherein at least 20 C-terminal residues of the yeast Ga subunit are replaced with corresponding C-terminal residues of the mammalian Ga subunit such that the chimeric Ga subunit is capable of being activated by the mammalian GPCR.
- the yeast periplasmic display library of embodiment 151 wherein the mammalian Ga subunit is selected from the group consisting of G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q, G alpha-l 1, G alpha-l2, G alpha-l3, and transducin.
- the yeast periplasmic display library of embodiment 156 wherein the human GPCR is selected from the group consisting of CXCR4, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB2, A3AR, and AT1R.
- ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of the GPCR.
- the antibodies are selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, recombinant fragments of antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, F v fragments, and scFv fragments.
- yeast periplasmic display library of embodiment 133 wherein the yeast host cell is a ⁇ far 1. ⁇ sst2. ⁇ ste 14. ⁇ stc3. or ⁇ mat strain.
- the yeast periplasmic display library of embodiment 163 wherein the ⁇ mat strain comprises a deleted or inactivated MATa locus or a deleted or inactivated MATa locus.
- yeast periplasmic display library of embodiment 133 wherein the yeast host cell further comprises a modified CLN3 protein comprising a C-terminal truncation that increases abundance of CLN3 in the yeast host cell compared to a wild-type CLN3 protein.
- CLN3 protein retains at least N-terminal amino acids 1-387 of the wild-type CLN3 protein.
- CLN3 protein retains at least N-terminal amino acids 1-408 of the wild-type CLN3 protein.
- yeast periplasmic display library of embodiment 133 wherein the yeast host cell is a haploid or diploid yeast host cell.
- the yeast periplasmic display library of embodiment 170 wherein the membrane associated protein domain is a glycosylphosphatidylinositol (GPI)-plasma membrane anchoring domain.
- GPI glycosylphosphatidylinositol
- the yeast periplasmic display library of embodiment 172 wherein the yapsin GPI plasma membrane anchoring domain is a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 yapsin GPI plasma membrane anchoring domain.
- the periplasm anchor protein is a protein that binds to an inner face of the cell wall such that the displayed protein variant is projected into the periplasm.
- each recombinant polynucleotide encodes a different fusion protein comprising the periplasm anchor protein linked to a different antibody for display;
- yeast host cells under conditions that permit expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest localizes to the plasma membrane, such that the yeast periplasmic display library of embodiment 134 is produced.
- the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.
- the method of embodiment 180 further comprising introducing into the plurality of yeast host cells a recombinant polynucleotide encoding an engineered Ga subunit capable of being activated by the GPCR, wherein the activated engineered Ga subunit is capable of activating a detectable pheromone response in the yeast host cell.
- the engineered Ga subunit is a chimeric G protein alpha (Ga) subunit comprising an N-terminal domain of a yeast Ga subunit and a C-terminal domain of an exogenous Ga subunit.
- yeast Ga subunit belongs to a Gai, Gaq, Gas, or Gao family G protein.
- yeast host cell is a FAR1 strain for selection of antibody antagonists of the target GPCR of interest.
- yeast host cell is a ⁇ farl strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of antibody agonists of the GPCR.
- a method of screening the yeast periplasmic display library of embodiment 136 for an antibody that modulates activity of the target membrane protein of interest comprising culturing at least a subset of the yeast host cells of the yeast periplasmic display library of embodiment 136 in a selection media; and detecting expression of the reporter gene, wherein increased expression of the reporter gene indicates that the antibody increases activity of target membrane protein of interest and decreased expression of the reporter gene indicates that the antibody decreases activity of the target membrane protein of interest.
- reporter gene is a nutritional marker, antibiotic resistance marker, fluorescent marker, biolumine scent marker, or a counter-selectable marker.
- embodiment 190 further comprising performing positive selection for expression of the nutritional marker, wherein growth of the yeast host cells in a nutrient-deficient selection media indicates the target membrane protein of interest is activated.
- antibiotic resistance marker confers resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.
- embodiment 190 further comprising performing positive selection for expression of the fluorescent marker, wherein detection of fluorescence emitted by the yeast host cells indicates the target membrane protein of interest is activated.
- the fluorescent marker is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, and an orange fluorescent protein.
- biolumine scent marker is luciferase or aequorin.
- a method of screening the yeast periplasmic display library of embodiment 148 for an antibody that modulates the activity of the target GPCR of interest comprising culturing at least a subset of the yeast host cells of the yeast periplasmic display library of embodiment 148 in a media, wherein detection of activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell not having an antibody displayed in the periplasmic space indicates that the displayed antibody in said at least one yeast host cell binds to and modulates the activity of the GPCR.
- yeast host cell is a ⁇ far l strain comprising a pheromone -inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds to and activates the GPCR in the yeast host cell results in increased expression of the reporter gene.
- reporter gene is a nutritional marker, antibiotic resistance marker, fluorescent marker, biolumine scent marker, or a counter-selectable marker.
- GPCRs G-protein coupled receptors
- GPCR ligand-binding pockets potentially causing significant off-target side effects.
- antibodies and related affinity molecules e.g., nanobodies and ScFvs and Fabs
- antibodies can better interact with extracellular domains and loops, which can modulate the structure (and thus function) of GPCRs in more sophisticated ways than small molecules.
- Our innovation includes combining GPCR-to-yeast pheromone response coupling and expressing affinity molecules that act in cis in the same cell in a high-throughput platform. This enables direct and high-throughput functional selection of affinity molecules (FIG. 1). To do this, we combine two strategies that are optimal for yeast:
- Yeast are the system of choice for expressing full length, functional GPCRs from humans for in vitro biochemical and structural studies. Remarkably, yeast have also been used successfully to functionally couple human GPCRs to the yeast pheromone response pathway and, depending on the readout, screen/select for small molecule, peptide, or protein ligands that functionally interact with a GPCR (King 1990, Brown 2000, Erlenbach 2001, Minic 2005, Dowell 2009, Dong 2010, Liu 2016).
- the most "universal" method of coupling is to modify the yeast Ga subunit, Gpal, to bind to a human GPCR and transduce signals by transplanting the 5 C-terminal residues of the cognate human Ga to replace the native 5 C-terminal amino acids in Gpal to make a "Ga transplant” (Conklin 1993, Brown 2000, Erlenbach 2001).
- Other methods include complete replacement of Gpal with the full-length cognate Ga of the human receptor (King 1990), or construction of a more complex Gpal/Ga chimera, with different portions of each combined into a hybrid human/yeast Ga (Klein 1998, Price 1995).
- affinity molecules can functionally interact in cis with GPCRs in the membrane.
- GPI-anchoring domain of YPS1 Frieman 2003
- Haploid yeast of mating type "a” undergo cell cycle arrest when the mating pheromone, alpha factor (a factor) activates its cognate GPCR receptor Ste2. This growth-arrest phenotype can be used in selection of Ste2 antagonists.
- spontaneous "pheromone resistant" mutants arise at a staggering rate.
- the background or "false positive" rate in our parental haploid strain is -10 4 , i.e., -100 colonies grow when 10 6 cells are spread in a- factor plates.
- we engineered a pheromone-responsive diploid base strain (Herskowitz 1989). False positives caused by loss-of-f mction mutations appear much less frequently in diploids.
- Some constitutively-expressed yeast genes depend on the basal activity of the pheromone response pathway to be expressed.
- We constructed our selectable marker by placing the promoter of one such gene, MFA1, driving the expression of HIS3, a gene required for cells to synthesize its own histidine. (Daniel 2006).
- MFA1 driving the expression of HIS3
- H- media histidine
- the engineered diploid strain (NIY326) carrying P(MFAl)-HIS3 and plated in H- media with a- factor had a background rate of 10 7 .
- Table 1 Summary of strain development to reduce background rate. Background is colonies formed per 10 7 cells plated on pheromone-containing agar
- periplasm-targeting expression vectors driving expression of a chimera with an N-terminal secretion signal, MFalpha PrePro (Brake 1984), followed by an 18- amino-acid linker containing a single FLAG epitope (DYKDDDDK) and ending in the YPS1 glycophosphatidylinositol (GPI) anchoring domain.
- the PrePro signal targets the protein for translocation into the endoplasmic reticulum and secretion, and is later cleaved off, while processing of the YPS1 GPI domain in the ER results in an N-terminal GPI anchor that retains the chimera tethered to the plasma membrane (Frieman 2003).
- the vectors carry the selectable marker URA3, which allows for positive selection in uracil-deficient media and also for counterselection (see below).
- MATalpha cells express the a-factor receptor Ste3 and do not express Ste2.
- Ste3 is a GPCR only distantly related to Ste2
- the a-factor pheromone is a glycosylated peptide entirely different from a-factor.
- the pheromone signaling cascades of MATa and MATalpha downstream of the receptor are identical. Both MATa and MATalpha cells arrest their cell cycle and activate pheromone-inducible genes in response to their cognate pheromone.
- MATa and MATalpha tester cells carry a pheromone-inducible transcriptional reporter, P(PRMl)-YFP that can be used to test pathway function in the absence and presence of the antagonist candidates.
- P(PRMl)-YFP pheromone-inducible transcriptional reporter
- the recombinant nanobodies with a fluorescent dye (Alexa 488, compatible with GFP wavelengths; Kit #A20l8l, Thermo-Fisher Scientific) and test their ability to stain the plasma membrane of Ste2-expressing cells and not control Ste3 -expressing cells.
- the immunofluorescence experiments also allow us to compare the staining of fixed cells with and without their cell walls (which can be easily removed with a lyticase enzymatic treatment), and thus confirm that the nanobodies can diffuse effectively through the cell wall. Finally, immunofluorescence is used to confirm that antagonist candidates directly bind the receptor rather than the ligand to exert their effects.
- HIS3 strains is much lower than for the antagonist selection strain because mutations that turn on the pheromone cascade are rarer than those that turn it off (Brown 2000).
- mutations that turn on the pheromone cascade are rarer than those that turn it off (Brown 2000).
- yeast clones expressing candidate agonist nanobodies for Ste2 Similar to the antagonist screens, we select yeast clones expressing candidate agonist nanobodies for Ste2 based on growth in H- plates. We test whether growth is plasmid/expression dependent and confirm specificity by testing hits in the Ste3 agonist selection strain (using the P(PRMl)-HIS3 and P(PRMl)-YFP reporter). Also, as above, we produce the nanobodies in bacteria and add them directly to cells to test their effectiveness.
- a widely used method for coupling human GPCRs to the yeast pheromone pathway is to modify the yeast Ga, Gpal, such that its 5 C-terminal amino acids are changed to those of a human Ga, generating a "GPA1 transplant” (Brown 2000, Erlenbach 2001).
- the suitable Ga is often found empirically (Dowell 2009).
- coupling is achieved with transplants for either Gori, Gaq, Gas or Gao (Dong 2010).
- CRISPR approach Horwitz 2015
- nanobodies (15 kDa) can diffuse through the yeast cell wall (Ries 2012), larger ScFvs (27 kDa) might be significantly constrained.
- CB2 receptor human cannabinoid receptor type 2
- VHH domain is presented by 1) a short linker connected to a Ypsl plasma membrane anchor 2) a long linker connected to a Ypsl plasma membrane anchor 3) an N-terminal fusion connected to the soluble periplasmid enzyme Suc2 and 4) direct secretion of untagged VHH into the periplasm. From two to five independent clones were grown to saturation in the absence of selection.
- Each of the four agonist VHH expression plasmids increased the growth rate in the second culture media formulation compared to cells transformed with empty plasmid (no VHH).
- the CB2 receptor can be activated by VHH domain antibodies that are covalently connected to a plasma membrane anchor protein, whether through a long or a short linker.
- VHH domain agonist antibodies can be localized to the periplasm by fusing the antibody to a periplasmic protein, Suc2, that is sufficiently large such that the fusion protein is retained in the periplasm.
- Suc2 forms oligomers comprising multiple Suc2 proteins linked by non-covalent interactions and this multimerization is required for retention of Suc2 in the periplasm. Therefore, this condition also demonstrates retention in the periplasm partly through non-covalent interactions between the antibody and the anchor protein.
- this experiment demonstrates that direct secretion of untagged VHH domain antibodies into the periplasm can activate the CB2 receptor.
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